The chemical synapse is highly specialized to perform efficient synaptic transmission. These synaptic specializations are localized to discreet regions of the neuron and occur in register between the synaptic partners. The long-term goal of this proposal is to understand the molecular mechanisms which allow nerve cells to form and maintain specific, spatially localized, synaptic connections. Toward this long term goal, a technique of patterning proteins on glass coverslips (microcontact printing:uCP) will be employed to covalently immobilize proteins in micron-scale patterns on glass coverslips suitable for neuronal cell culture. Cerebellar granule cells cultured on these substrates will be assessed immunologically, ultrastructurally and functionally for synaptic differentiation. This approach is ideally suited for identifying and characterizing molecules involved in synapse formation and maintenance for several reasons. 1. Synaptic differentiation is induced without a postsynaptic cell affording a "clean" preparation. 2. Micron-scale patterns are appropriate for synapses. 3. The parallel nature of the immobilization allows multiple proteins to be patterned on single coverslips at discrete, defined, locations enabling high throughput assays. The specific aims are 1. to use the synaptogenic activity of neuroligin to validate the uCP method of patterning proteins for assays of neuronal synaptic differentiation, and 2. to demonstrate the feasibility of using uCP to assess synaptogenic activity in mixtures of proteins. These proposed experiments are ideally suited to the exploratory /development grant program since they will validate a novel method for investigating interneuronal synaptogenesis. Development of this method would provide a powerful functional assay which can directly assess the ability of identified extracellullar proteins to induce synaptic differentiation, and provide a technique for identifying novel extracellular cues with local differentiation-inducing activity. Application of the methods developed in this proposal will greatly enhance our understanding of molecular mechanisms of synapse formation and maintenance: processes with important implications for all central nervous system functions which rely on stability or plasticity of synaptic contacts.